Dendritic cells (DCs) were derived from adherent PBMCs and cultured in CellGro DC medium (CellGenix, Freiberg, Germany) in the presence of interleukin (IL)-4 (1,000 U/mL) and granulocyte macrophage colony-stimulating factor (GM-CSF; 800 U/mL; both R&D Systems, Minneapolis, MN). On day 5, immature DCs were matured for 48 hours using a cytokine cocktail consisting of IL-4, GM-CSF, IL-6 (10 ng/mL), TNF-α (10 ng/mL), IL-1β (10 ng/mL; all R&D Systems), and PGE2 (1μg /mL; Sigma-Aldrich, Hayward, CA).
Activated T-cells (ATCs) for use as APCs were generated from PBMCs by stimulation on non-tissue-culture-treated 24-well plates coated with 1 μg/mL OKT3 (Ortho Biotech, Bridgewater, NJ) and 1 μg/mL anti-CD28 (Becton Dickinson, Franklin Lakes, NJ) antibodies in T-cell medium (50% RPMI 1640 [Hyclone], 45% EHAA [Life Technologies, Santa Ana, CA], 2 mM Glutamax and 5% human AB serum [Valley Biomedical Inc, Winchester, VA]) in the presence of human IL-2 (50 IU/mL, from the NCI Biological Resources Branch, Frederick MD) for 8 to 14 days. ATCs were restimulated with CD3 and CD28 antibodies to upregulate HLA and costimulatory molecules 2 days prior to use as APCs [26 (link)].
Activated T-cells (ATCs) for use as APCs were generated from PBMCs by stimulation on non-tissue-culture-treated 24-well plates coated with 1 μg/mL OKT3 (Ortho Biotech, Bridgewater, NJ) and 1 μg/mL anti-CD28 (Becton Dickinson, Franklin Lakes, NJ) antibodies in T-cell medium (50% RPMI 1640 [Hyclone], 45% EHAA [Life Technologies, Santa Ana, CA], 2 mM Glutamax and 5% human AB serum [Valley Biomedical Inc, Winchester, VA]) in the presence of human IL-2 (50 IU/mL, from the NCI Biological Resources Branch, Frederick MD) for 8 to 14 days. ATCs were restimulated with CD3 and CD28 antibodies to upregulate HLA and costimulatory molecules 2 days prior to use as APCs [26 (link)].
